Method for producing biomaterials from bone tissue and material used for osteoplasty and tissue engineering

ABSTRACT

A method for obtaining of biomaterials for osteoplasty and tissue engineering cleans a bone of natural origin, which is sawn to plates with a thickness of 0.2 to 2.0 cm, washed twice in 0.1 M phosphate buffer at 65° C., pH 5.8-6.0, calculated as two volumetric parts of the buffer solution per one part of bone, digested in a solution of activated 0.1-0.4% papain at 65° C. for 24 hours. The plates are washed with 5 volumes of water at the temperature 40-80° C., treated with 0.4 N alkali solution at room temperature for 10-24 hours, washed with flowing water, dried, defatted in ethanol/chloroform mixtures in proportion of 1:2 and then in proportion of 2:1, decalcified in 0.4-1 N hydrochloric acid, treated with 1.5-3% hydrogen peroxide for 4 hours, washed with treated water, then washed with ethanol, dried at room temperature, packed and sterilized.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of international application numberPCT/RU2005/000526, filed on Oct. 27, 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention covers the field of medicine, in particular, biochemistryand biomaterials extraction technology and is also used formanufacturing of biomaterials applied as a plastic material by theoperative restoration of bone defects by destruction of bone tissue,cystectomy, oncotomy and also as a carrier of active substances anddrugs, in plastic surgery by restoration of organ's or tissue's volume.

2. Description of the Related Art

A bone is a living tissue, in which a continuous reorganization processincluding a simultaneous destruction and restoration of the bonematerial. Old tissue is remodulated during a normal process and also byimplantation of a foreign material, new tissue is formed in place ofthis. Equilibrium is permanently maintained between the quantity of areformed bone and a newly developed bone. This process will go easier ifthe implanted material is close to a habitual bone in its structure.

That is why at present it is preferred to prepare the substitutematerial from tissues of a natural bone, which must be of animal originby reasons of ethics and practice.

It is well-known that a partial demineralization promotes implantationof a bone graft. Hereupon various additional steps are taken, which areintended either for full deproteinization of the bone or for influencingthe nature of proteins remaining bound in the bone base or for increaseof this share of proteins.

As regards to methods used heretofore, in particular, the U.S. Pat. No.4,394,370 can be given as an example, where it is proposed to form aspongy mass by the melting of a mixture consisting of a powder of thedemineralized bone of human origin and a diluted collagenic powder withthe help of glutaraldehydic binding providing a cross-link.

The U.S. Pat. No. 4,743,259 combines demineralization by hydrochloricacid with enrichment by proteins carried out on the first part of thedemineralized bone with the help of proteins extracted from the secondpart with use of guanidine.

Furthermore, it is proposed in patent application FR No. 2582517 toprocess bone ends taken from animals, namely, from livestock by means ofa partial demineralization and tanning with use of glutaraldehyde. Bonefragments to be implanted by a surgeon are cut out with necessaryshaping from cattle's bones after a preliminary pretreatment includingdelipidization or defatting operation with the use of an organic solventsuch as ethanol, demineralization with the use of calcium extractionmeans such as hydrochloric acid and an operation providing for tanningwith glutaraldehyde and also various washing operations.

It is obvious from the description of the prior art patents given abovethat the mentioned tanning process favors the features of a treatedbone, as it facilitates the cross-linkage of macromolecular chains.However, it has been recently detected that the treatment ofglutaraldehydes does not result in a significant reduction ofimmunogenic features and, moreover, the engraftment of the implantedbone occurs in a desirable degree, as opposed to the theory suggestedearlier. Furthermore, such chemical compounds like glutaraldehyde have adisadvantage in that they are biologically toxic.

A method for obtaining a material for osteoplasty from the bone tissueof natural origin including the subsequent removal of lipids from thenatural bone tissue with use of an organic solution, selectiveextraction with the subsequent washing and lyophilization of the endproduct is known, characterized by the selective extraction carried outwith the use of a urea solution for denaturation and removal ofantigenic proteins with the maintaining of a non-denaturated collagen oftype I in a natural form located in the initial mineral bone structureand the obtained structure is directed to washing and lyophilization (RUNo. 2104703, A61K35/32, publ. Feb. 20, 1998).

Therefore, the removal of lipids is carried out with use of the organicsolution containing 10 volumetric parts of chloroform/methanol orethanol/dichloromethane mixture per 1 part of bone in proportion 2:3-1:3accordingly. The demineralization of the bone tissue is carried out withthe solution of the hydrochloric acid with molarity of 0.1-1.0 M afterremoval of lipids. The extraction with an ionic solvent is carried outbefore the selective extraction, in particular, with use of sodiumchloride.

The selective extraction is conducted with a 2 to 10 M urea solution,preferably with a 5 to 8 M solution or aqueous urea solution containing0.1-0.5 vol. of mercaptoethanol. The washing is carried out with use ofdistilled water at 30-60° C., preferably at 45-55° C. Alternatively, theselective extraction is carried out first with use of the urea solutionin concentration from 2 to 10 M, preferably within the limits of 5 to 8M, then, after washing, with use of the aqueous urea solution containingmercaptoethanol in quantity of 0.1 to 0.5 vol. in the solution.

The material for osteoplasty obtained by such a way represents acompound, in which the bone structure of natural origin containing20-40% (namely 25-35%) of a non-denaturated collagen of type I ismaintained. According to analysis results of dry material, this materialcontains less than 15% lipids, 25-45% proteins, 10-30% calcium, 5-20%phosphorus and has a water content below 10% and a Ca/P ratio inpreference from 1 to 2.2.

The material can have a shape of parallelepipedic blocks, truncatedpyramids, plates, discs or powder amalgamated with a binder which canpreferably be of biological origin such as fibrin or synthetic originsuch as, for example, synthetic biodegradable polymer.

This invention is chosen as a prototype both for the method andmaterial, as it is the closest to the proposed invention as regards toits engineering solution.

The disadvantage of the mentioned method is the fact that suchtreatment, although it maintains a bone collagen of type 1 in thenatural form, does not provide full removal of antigens such asnon-collagenic proteins, lipids, lipoproteins and other substancesreducing the biocompatibility of the obtained material from this tissue.

BRIEF SUMMARY OF THE INVENTION

The goal of the invention is to improve the quality of the biomaterialobtained containing hydroxyapatite and/or bone collagen from a bonetissue and to obtain materials for application in stomatology,traumatology and orthopedics on its basis by means of maintaining thenative structure of the bone collagen and spatial organization of thebone tissue for its subsequent cellular colonization, engraftmentcapacity of such biomaterials at the expense of their antigenicfeatures, to increase the biocompatibility and biointegration.

The technical result reached by use of this invention is the obtainingof a bone biomaterial with maintained architectonics and pure bonecollagen being low-antigenic materials, which can be widely used forobtaining products of medical purpose, such as materials for restorationof bone defects and also as a carrier of active substances and cells andwhich is able to be a base for other products of medical purpose.

As regards to the method, the mentioned technical result is reached inthat the bone is sawn to plates with the thickness of 0.2 to 2.0 cm,washed with 0.1 M phosphate buffer, pH 5.8-6.0, heated to 65° C.,digested in the solution of the activated 0.1-0.4% papain at 65° C. for24 hours, then the plates are washed with 5 vol. parts of water at40-80° C. (preferably 50-60° C.), treated with the 0.4 N alkali solutionat room temperature for 10-24 hours, washed with flowing water, defattedin ethanol/chloroform mixtures in proportion of 1:2 and 2:1, decalcifiedin 0.4-1N hydrochloric acid, treated with 1.5-3% hydrogen peroxide for 4hours, washed with treated water, then with ethanol at room temperature,and packed and sterilized.

As regards to the material, the technical result is reached in that thematerial for osteoplasty and tissue engineering obtained on the basis ofthis method represents a compound with the native spatial organizationof the collagenic matrix and mineral component of the bone tissue ofnatural origin containing 25% of collagen and 75% of mineral substance.According to analysis results of the dry material, this materialcontains less than 1% of non-collagenic proteins.

A spongy or cortical bone of human or vertebrates, e.g., pigs, bucks,hens, geese etc. can serve as a material for obtaining the bone collagenand products on the basis of this. This tissue mainly consists ofcollagen of type I and III and is characterized by low solubility andalso high resistance to collagenase. This type of the collagen is themost widely used in products for medical purposes being implanted intothe organism tissues.

The attributes mentioned both for the method and for the material areessential and interconnected with forming a stable aggregate ofessential attributes sufficient for obtaining the required technicalresult.

The technical essence of the method according to this invention andcharacteristics of the material for osteoplasty and tissue engineeringobtained with use of this method are described below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to international application numberPCT/RU2005/000526, filed on Oct. 27, 2005, which is incorporated hereinby reference in its entirety.

The process of preparation of bioimplants applied according to thismethod requires an initial mechanical treatment of the tissue, when thebone is cleaned free from any remaining soft tissues and blood.

An essential attribute of the invention is the procedure of the bonetreatment. After mechanical treatment the tissue is sawn up to formplates with a thickness not less than 0.2 cm and not more than 2.0 cm,as these dimensions are optimal by treatment of this tissue withsolutions. The minimum size of plates with the minimum thickness 0.2 cmand minimum thickness 2.0 cm were determined by us experimentally. So,difficulties emerge with accessibility of enzyme and other solutions toactive places of the substrate and also by washing of such plates freefrom solutions applied according to this technology by the increase ofthe plate thickness. Serious problems with maintaining continuity of thebone collagen and spatial structure of the bone tissue during thematerial treatment process arise by reduction of the plate thickness.

After cutting, the tissue is washed with the double volume of the 0.1 Mphosphate buffer with pH 5.8-6.0 heated to 65° C. Namely washing in thephosphate buffer heated to 65° C. precedes digesting with the enzyme andcreates optimal conditions for subsequent effect of the papain enzyme bysufficient reduction of incubating time with the enzyme by these pHvalues.

The enzyme is able to destroy non-collagenic proteins, proteoglycans andglycoproteins of the bone tissue effectively under these conditions,while fibers of the bone collagen are fully screened with ahydroxyapatite layer and so the bone collagen fibers are not exposed todenaturation and destruction maintaining their native structure bydigesting in conditions of increases of temperature up to 65° C. This isclearly seen on the output of sulphated glycosaminoglycans and aminoacids to digest.

Various concentration of the enzyme can be taken depending on thestructure of the bone tissue and its thickness. So, the concentration of0.1% activated papain is sufficient by the thickness of the spongy boneof 0.2 cm or, in case of treatment of the cortical bone, the papainconcentration is increased to 0.4%.

The optimal effect of the papain onto the bone tissue by digesting ofproteins and proteoglycans is 24 hours at 65° C. At that time, themaximum quantity of non-collagenic proteins and proteoglycans is removedfrom the bone tissue. It has been found by us experimentally that about2 g of glycosaminoglycans is extracted to the digest from 1 kg of bonesin 24 hours, which is practically equal to the theoretically calculatedquantity of sGAG for this type of tissue (Chvapil M., Physiology ofconnective tissue, Butterworths, London, 1967, p. 67-70).

The bone plates are washed with 5 vol. parts of flowing water heated to40-80° C. (preferably 50-60° C.) after their digesting with the enzyme.This operation allows for removal of all reaction products of thesubstrate with the enzyme, the enzyme itself and the major part of fats(more than 90%).

The effective defatting and removal of possibly remaining non-collagenicproterins is reached by the effect of the alkali onto thenon-decalcified bone tissue. The treatment of the bone tissue with 0.4HNaOH (sodium hydroxide) is carried out for 10-24 hours at roomtemperature. It is well-known that this alkali is a very effective agentfor destructing protein compounds, bacterial and viral particles, withwhich the bone tissue can be contaminated. This stage must be carriedout at room temperature (18-20° C.), as the efficiency of such effectsis significantly reduced by lower temperatures and the structure of thecollagenic molecule itself and of the collagenic matrix can be destroyedby the temperature increase. As in the case with the enzyme, the initialmineral bone structure covering the collagenic matrix of the bone doesnot allow the 0.4H alkali solution to have an effect on the structure ofthe bone collagen even after its influence for 24 hours.

The spongy bone with the plates having sizes of 0.2 to 0.5 cm is treatedfor 10 hours, as protein molecules located on the surface of thecollagen fibers are fully destroyed during this period of time. Thethicker plates of the spongy bone and fragments of the cortical bonerequire the application of an alkali for 24 hours. The protein is notdetected in the washouts from the tissue after this time.

After treatment with alkali, the bone is washed 5 times in flowing waterand the plates are dried at room temperature.

As opposed to all known methods of obtaining biomaterials from the bone,this method proposes to carry out initial treatment of washing andenzymatic influence at high temperatures (65° C.), but then thedestruction of the collagen molecule and collagenic matrix as a wholedoes not occur. In addition, it is proposed in this method to proceedwith defatting and decalcification of the bone after its treatment withthe enzyme and alkali only, as main antigens are already removed fromthe non-decalcified bone and the bone collagen remains practicallyunchanged due to a protective layer of hydroxyapatite covering the bonefiber.

A significant quantity of fats and their compounds with proteins andcarbohydrates is contained in the bone tissue. The lipids are both inthe free state and in form of compounds with sugars such aslipopolysaccharides, which are active antigens and can cause variousinflammatory complications in the bone plates treated with the enzyme.The method includes the treatment of bone plates in mixtures ofchloroform and ethanol in proportions of 2:1 and 1:2 at the stage, whenthe main bone stroma is already cleaned free from its other components,namely for removal of all remained lipids. The treatment in the mixtureis carried out for 48 hours in each mixture until the complete removalof lipids, which can be estimated on the basis of the content of fats in1 g of the dry tissue.

This stage ensures the liberation of lipids even from the dense bonetissue (cortical bone). After such treatment, their output is terminatedand the content in the material does not exceed 1%. After defatting, thebone plates are dried and decalcified in solutions of mineral acids. Asa rule, thin plates of the spongy bone are treated with a 0.4H solutionof hydrochloric acid (HCl), and thicker plates (1 cm and more) in 1H HCland the process is continued until the complete elimination of Ca⁺⁺ ionsin the decalcifying solution. The decalcification process of the bonemineral cannot be conducted at all or the demineralization degree of thematerial can be strictly programmed.

The analytical study of the method for obtaining a material withoutdemineralization has shown that the obtained material has classicindications of the bone tissue: 25% of collagen and 75% of mineralsubstance. At that, not only the structural collagen is not affected,but the spatial organization of the collagenic matrix and mineralcomponent part of the bone tissue remains fully maintained. The obtainedmaterial differs from all materials at present applied in osteoplasty,both in its composition and operating characteristics and also in fullabsence of non-collagenic components and antigenic features. Thismaterial practically fully maintains the native spatial structure of thebone tissue, which is particularly necessary for good integration,biocompatibility and cellular colonization. Furthermore, this materialundergoes treatment with 1.5-3% hydrogen peroxide for 4 hours.

This stage allows, first, the removal of remaining non-collagenicprotein molecules, and second to destroy a series of other compoundssuch as pigments, remained lipids, not readily soluble salts, etc. 3%hydrogen peroxide is usually used for treatment of plates with athickness of more than 1.0 cm. The obtained collagen is then washed 5times in treated water, then in ethanol, dried at the room temperature,packed and sterilized.

The obtaining of the material is controlled at each processing stage andincludes main methods accepted for these types of materials.

The absence of proteoglycans was determined on changes in coloration ofthe substrate and in the solutions spectrophotometrically in thepresence of 1.9-dimethylene blue by the wavelength of 535 nm based onFarndel's method.

The protein output was determined based on Lowry's pharmacopeia methodspectroscopically by the wavelength of 400 nm and the presence ofcollagen remainders in washouts with the use of Kjeldal's method fordetermination of oxyprolines.

The presence of calcium ions in decalcifying solutions was determinedwith the help of a qualitative reaction to Ca²⁺. The control of lipidswas carried out by painting the material with Sudan. The structuralintegrity of the bone collagen was determined by study of histologicsections, electron microscopically and with use of scanning microscopymethod. It was established with the help of these methods that theporous-fibrous structure of the bone collagen has a typical appearancewithout any changes and abnormalities.

The control instrumentation was carried out after drying andsterilization for measurement of the content of non-collagenic proteinsin the material as compared with the prototype. So, 4-5% of protein isdetermined per dry weight of the material obtained according to themethod described in the prototype and less than 1% according to themethod proposed in the present invention. Thus, the proposed methodallows for a significant reduction of the material's antigenicity due toa more complete removal of non-collagenic proteins as compared with theprototype. Therefore, the proposed method of tissue treatment maintainsthe native structure of the material, improves its quality, reduces thematerial's and thereby ensures good plastic features, biointegration andbiocompatibility.

Brief Description of a Process Used for Obtaining of Material

The pig's bone, passed through a required veterinary control, is cleanedof muscles and tendons, sawn up into plates with a thickness of 2.0 cmand put into a 0.1 M phosphate buffer at 65° C., pH 5.8-6.0. The bufferis poured off and the material is washed again with the heated bufferand transferred into a solution of activated 0.4% papain. The incubatingis carried out at 65° C. in the thermostat for 24 hours. The digest isthen poured off and the plates are washed with 5 volumes of water heatedto 70° C., cooled to room temperature and put into a 0.4H alkalisolution for 24 hours. The material is washed free from alkali, driedand treated twice, first with an ethanol/chloroform mixture inproportion of 1:2 for 48 hours and then with an ethanol/chloroformmixture in proportion of 2:1 for the subsequent 48 hours.

The bone plates are dried again and put into the 1 N hydrochloric acid.The change of acid is carried out until there is complete elimination ofcalcium ions in the decalcifying solution. The acid is washed with waterand the plates are put into 3% hydrogen peroxide for 4 hours. Then theplates are washed with the treated water and ethanol, the material isdried, packed and sterilized.

These actions mentioned above lead to reduction of antigenicity andmaintaining of the collagenic structure and bone collagenic matrix.

The quantitative and qualitative analysis of the bone collagen isconducted as described above.

The invention is explained by examples of specific executions for abetter insight of the present invention.

Example 1

A donor human bone, passed through the required analyses, ismechanically cleaned of muscles and tendons, sawn up into plates with athickness of 0.2-0.6 cm and put into a 0.1 M phosphate buffer at 65° C.,pH 5.8-6.0, twice for 30 minutes each time. The buffer is then pouredoff and the plates are transferred into a solution of activated 0.15%papain at 65° C. into the thermostat for 24 hours. Then the supernatantis poured off, the plates are washed with 5 volumes of water at 60° C.,cooled to room temperature and put into the 0.4H alkali solution for 24hours. The material is washed free from alkali and dried. The plates areput first into an ethanol/chloroform mixture in proportion of 1:2 twicefor 4 hours and then twice into the same mixture but in proportion of2:1 for 24 hours.

The material is dried and treated with the 1.5% hydrogen peroxide for 4hours. Then the plates are washed first with the treated water and thenwith ethanol. For obtaining a bone collagen, the material is dried afterits defatting in organic solutions and decalcified in 0.5 N hydrochloricacid. The acid is washed out and the bone plates are treated with the1.5% hydrogen peroxide for 4 hours. Then the material is washed againfirst with the treated water and then with ethanol. The material treatedby the use of such method is dried at room temperature, frozen-dried,packed and sterilized by use of a radiation method. Bones of animalswith a thickness of up to 0.8 cm are treated in a similar manner.

The analytic study with respect to the presence of proteins,proteoglycans and lipids in the material is conducted at the end of eachtechnological cycle. The obtained materials are applied for restorationof bone defects by operative intervention in stomatology, orthopedicsand traumatology. The non-decalcified bone material and bone collagencan be saturated with bioactive substances (sulfated glycosaminoglycans,growth factors such as PDGF, IGF, FGF etc.) for clinical, experimentaland scientific purposes and also can be used as carriers for varioustypes of cells such as stem cells, embryonic cells, blood cells etc.

Example 2

The pig's spongy bone, passed through a required veterinary control, ismechanically cleaned of muscles and tendons, sawn up into plates with athickness of 1.1-2.0 cm and put into a 0.1 M phosphate buffer at 65° C.,pH 5.8-6.0, twice for 60 minutes each time. The buffer is then pouredoff and the plates are transferred into a solution of activated 0.3%papain at 65° C. into the thermostat for 24 hours. Then the digest ispoured off, the plates are washed with 5 volumes of water at 70° C.,cooled and put into the 0.4H alkali solution at room temperature for 24hours. The material is washed free from alkali and dried.

The plates are put first into an ethanol/chloroform mixture inproportion of 1:2 twice for 4 hours and 24 hours and then twice into thesame mixture but in proportion of 2:1 for the subsequent 4 hours and 24hours. After defatting, the material is dried and treated immediatelywith an 3.0% hydrogen peroxide for 4 hours or decalcified in the 1 Nhydrochloric acid and then treated with the 3.0% hydrogen peroxide for 4hours with the following washing free from the acid. Both kinds ofmaterial are washed first with the treated water and then with ethanol.

The bone matrix and bone collagen obtained in such a way are cut intofragments of various shape and size: cubes, parallelepipeds, blocks etc.and dried at room temperature or by use of a lipophilic method, packedand sterilized by radiation. Bones of various animals and humans with athickness exceeding 1 cm are treated in a similar manner.

The analytical study with respect to the presence of proteins,proteoglycans and lipids in the material is conducted at the end of eachtechnological cycle. The obtained materials are applied for restorationof bone defects by operative intervention in stomatology, orthopedicsand traumatology. The non-decalcified bone material and bone collagencan be saturated with bioactive substances (sulfated glycosaminoglycans,growth factors such as PDGF, IGF, FGF etc.) for clinical, experimentaland scientific purposes and also can be used as carriers for varioustypes of cells such as stem cells, embryonic cells etc.

Example 3

The pig's cortical bone, passed through a required veterinary control,is mechanically cleaned of muscles and tendons, sawn up into plates witha thickness of 1.5-2.0 cm and put into a 0.1 M phosphate buffer at 65°C., pH 5.8-6.0, twice for 60 minutes each time. The buffer is thenpoured off and the plates are transferred into a solution of activated0.4% papain at 65° C. into the thermostat for 24 hours. Then the digestis poured off, the plates are washed with 5 volumes of water at 80° C.,cooled and put into a 0.4 N alkali solution at room temperature for 24hours.

The material is washed free from alkali and dried. The plates are putfirst into an ethanol/chloroform mixture in proportion of 1:2 twice for4 hours and 24 hours and then twice into the same mixture but inproportion of 2:1 for the subsequent 4 hours and 24 hours. As in theprevious example, the material is dried and treated immediately with the3.0% hydrogen peroxide for 24 hours (osteomatrix) or decalcified in the1 N hydrochloric acid and only then treated with the 3.0% hydrogenperoxide for 24 hours after washing free from the acid (bone collagen).Both kinds of material are washed first with the treated water and thenwith ethanol.

The biomaterials obtained by such a way are cut into fragments ofvarious shape and size: cubes, parallelepipeds, disks, blocks etc. anddried at the room temperature, packed and sterilized by radiation. Bonesof various animals and humans with a thickness exceeding 1 cm aretreated in a similar manner. The analytical study with respect to thepresence of proteins, proteoglycans and lipids in the material isconducted at the end of each technological cycle.

The obtained materials are applied for restoration of bone defects byoperative intervention in stomatology, orthopedics and traumatology. Thenon-decalcified bone material and bone collagen can be saturated withbioactive substances (sulfated glycosaminoglycans, growth factors suchas PDGF, IGF, FGF etc.) for clinical, experimental and scientificpurposes and also can be used as carriers for various types of cellssuch as stem cells, embryonic cells etc.

Example 4

A donor human bone, passed through the required analyses, ismechanically cleaned of muscles and tendons, sawn up into plates with athickness of 0.5-0.8 cm and is treated as per example 1 until thedecalcification stage. Then defatted bone plates are dried and treatedwith the 1.5% hydrogen peroxide for 6 hours. The plates are washed firstwith the treated water and then with ethanol. The material treated insuch a way is dried at room temperature, frozen-dried, packed andsterilized by radiation. Bones of animals with the thickness of 0.5 to1.0 cm are treated in a similar manner.

The analytical study with respect to the presence of proteins,proteoglycans and lipids in the material is conducted at the end of eachtechnological cycle.

Example 5

The pig's spongy or cortical bone, passed through a required veterinarycontrol, is mechanically cleaned of muscles and tendons, sawn up intoplates with a thickness of 1.0-2.0 cm and then treated as per example 2until the decalcification stage. Then defatted bone plates are dried andtreated with the 1.5% hydrogen peroxide for 6 hours. The plates arewashed first with the treated water and then with ethanol. The materialtreated by such a way is dried at the room temperature, frozen-dried,packed and sterilized ethanol. Bones of animals with a thickness of 0.5to 1.0 cm are treated in a similar manner.

INDUSTRIAL APPLICABILITY

This invention is industrially applicable, mastered in vitro, such thatthe laboratory results show the practical value of the obtained materialfor osteoplasty and tissue engineering.

1. A method for obtaining bone plates, from tissue of a bone, the methodcomprising: (a) cleaning the bone; (b) sawing the bone into plateshaving a thickness of 0.2-2.0 cm; (c) washing the plates twice in a 0.1M phosphate buffer solution having a pH of 5.8-6.0 and a temperature of65° C., wherein the ratio of bone plates to buffer solution is 1:2 byvolume; (d) immersing the plates in a 0.1-0.4% solution of activatedpapain at 65° C. for 24 hours; (e) washing the plates five times withwater having a temperature 40-80° C.; (f) treating the plates with 0.4 Nalkali solution at room temperature for 10-24 hours; (g) washing theplates with water; (h) drying the plates; (i) removing fat from theplates by washing with an ethanol and chloroform mixture, wherein theratio of ethanol to chloroform is 2:1 by volume; (j) removing fat fromthe plates by washing with an ethanol and chloroform mixture, whereinthe ratio of ethanol to chloroform is 1:2 by volume; (k) decalcifyingthe plates with a solution of hydrochloric acid; (l) treating the plateswith a solution of hydrogen peroxide for 4 hours; (m) washing the plateswith water at room temperature until the hydrogen peroxide is removedfrom the plates; (n) washing the plates with ethanol; (o) drying theplates; (p) packing the plates; and (q) sterilizing the plates.
 2. Amethod for obtaining bone fragments from tissue of a bone, the methodcomprising: (a) sawing the bone into fragments having a thickness of0.2-2.0 cm; (b) washing the fragments in a 0.1 M phosphate buffersolution having a pH of 5.8-6.0 and a temperature of 65° C.; (c)immersing the fragments in a solution of activated papain; (d) washingthe fragments with water; (e) treating the fragments with a 0.4 N alkalisolution at room temperature; (f) washing the fragments with water; (g)drying the fragments; (h) removing fat from the fragments by washingwith an ethanol/chloroform mixture; (i) decalcifying the fragments witha solution of an acid; (j) treating the fragments with hydrogenperoxide; (k) washing the fragments with water until the hydrogenperoxide is removed from the fragments; (l) washing the fragments withethanol; and (m) drying the fragments.
 3. The method of claim 1, whereinthe drying in step (g) includes freeze-drying.
 4. The method of claim 1,wherein the papain solution is 0.1-0.4% papain.
 5. The method of claim1, wherein the papain solution is at 65° C.
 6. The method of claim 1,wherein the immersing step lasts for about 24 hours.
 7. The method ofclaim 1, wherein step (d) is performed multiple times.
 8. The method ofclaim 1, wherein the water in step (d) has a temperature of 40-80° C. 9.The method of claim 1, wherein step (e) is performed for 10-24 hours.10. The method of claim 1, wherein the ethanol and chloroform mixture ofstep (h) has a 2:1 ratio by volume of ethanol to chloroform.
 11. Themethod of claim 10, wherein in step (h) the bone fragments are furtherwashed with an ethanol and chloroform mixture having a 1:2 ratio byvolume of ethanol to chloroform.
 12. The method of claim 1, wherein theacid in step (i) is hydrochloric acid.
 13. The method of claim 12,wherein the hydrochloric acid concentration is 0.4-1.0 N.
 14. The methodof claim 1, wherein step (j) is performed for 4 hours.
 15. The method ofclaim 1, wherein the water in step (k) is at room temperature.
 16. Themethod of claim 1, further comprising the steps of: (n) packing thefragments; and (o) sterilizing the fragments.
 17. The method of claim 1,wherein the fragments are any of plates, cubes, parallelepipeds andblocks.
 18. The method of claim 1, further comprising cleaning the boneprior to step (a).
 19. The method of claim 1, wherein in step (b) theratio of bone fragments to buffer solution is 1:2 by volume.